Compositions comprising hydroxytyrosol and boswellic acid

ABSTRACT

Compositions are described including a synergistic combination of hydroxytyrosol and 3-O-acetyl-11-keto-β-boswellic acid. The hydroxytyrosol may be sourced from an olive extract and the 3-O-acetyl-11-keto-β-boswellic acid may be sourced from a  Boswellia serrata  extract. The compositions may be formulated for oral administration to a mammalian or an avian subject. Methods for treating, repairing, or reducing damage to connective tissue caused by one or more inflammatory mediators and for reducing levels of one or more inflammatory mediators in connective tissue are provided, the methods comprising orally administering the compositions to a subject in need thereof.

This application claims the benefit of priority of U.S. Continuationpatent appl. Ser. No. 15/494,022 filed on Apr. 21, 2017 which in turnclaims priority to PCT Patent Application Serial No. PCT/US17/28857filed on Apr. 21, 2017, which in turn claims priority to U.S.Provisional Patent Application Ser. No. 62/403,807 filed on Oct. 4,2016, the entirety of the contents of each of which are incorporated byreference herein.

TECHNICAL FIELD

The present invention provides methods comprising administration of: (i)3-O-acetyl-11-keto-β-boswellic acid (AKBA) and (ii) hydroxytyrosol, to amammalian or an avian subject. The present invention also providesorally administrable compositions comprising AKBA and hydroxytyrosol.

BACKGROUND

Connective tissue is the structural framework of cartilage, bone,synovium, ligament, meniscus, and tendon in articulating joints.Components of connective tissue are produced by resident cells and thensecreted to form the extracellular matrix (ECM) characteristics of thetissue. In addition to serving as structural framework, the ECM alsoplays a critical role in cell communication and function. In articularcartilage, chondrocytes are aligned in a distinct pattern within thetype II collagen ECM framework. Bone forming osteoblasts and osteocytes,as well as bone resorbing osteoclasts, are organized in mineralized typeI collagen ECM. The few fibroblast-like and macrophage-like cells in thesynovium are also held in place by ECM. Similarly, tenocytes andligament cells are assembled together within the ECM. The synthesis andbreakdown of connective tissue ECM is controlled by a network ofregulatory molecules which are also produced by the resident tissuecells. This network includes growth factors and a wide array ofmolecules known as pro-inflammatory mediators.

They include cytokines, chemokines, prostaglandins and nitric oxide.These molecules exhibit many biological activities. They can induce cellproliferation or cell death. These substances can also induce anabolicpathways for production of ECM or induce catabolic enzymes that canbreak down the ECM. Under physiological conditions, cell survival ordeath, the production or breakdown of connective tissue ECM is tightlycontrolled to maintain balanced homeostasis. The production and functionof regulatory molecules is modulated by many factors includingmechanical forces, physical factors such as temperature and pH,chemicals, microbes and their products. Under certain conditions, thesefactors can elicit excessive and untimely production of regulatorymolecules leading to irreparable tissue damage, loss of function anddeath.

Tissues react to mechanical, physical, chemical insults and infection byan inflammatory response. The inflammation process is known to lead torecovery, to healing, defense against infection and is usually lifepreserving. The inflammatory response in humans and animals consists oftwo phases. The initial phase is characterized by the local synthesis ofpro-inflammatory mediators such prostaglandins and leukotrienes. Theyare derived from arachidonic acid through the action of cyclooxygenasesand lipoxygenases. These pro-inflammatory mediators increase local bloodflow and enhance the permeability of endothelial cells to allowleukocyte recruitment and accumulation. Other pro-inflammatory mediatorswhich are subsequently produced include cytokines (Interleukin-1 beta(IL-1 β), tumor necrosis factor alpha (TNF-α)), chemokines (IL-8), andnitric oxide. In the second phase, the resolution phase, prostaglandinsgenerated during the initial phase activate enzymatic pathways alongwhich arachidonic acid is converted to chemical mediators withanti-inflammatory properties. It has been reported that prostaglandin E₂(PGE₂) activates the expression of 15-lipoxygenase which generatesanti-inflammatory lipoxins from arachidonic acid. Thus, the resolutionof inflammation is driven by the pro-inflammatory response. Thesestudies indicate that the initiation, progression and termination of theinflammation process are tightly controlled. Prolonged, exaggeratedinflammation has been associated with many disorders includingosteoarthritis (OA), rheumatoid arthritis (RA), Alzheimer's disease, andcardiovascular disease.

In joint tissues, chondrocytes, synoviocytes, osteoblasts, osteoclasts,ligament cells, and tenocytes produce a wide array of pro-inflammatorymediators. Among these is PGE₂, which is known to play a regulatory roleby inducing the production of other mediators including cytokines,nitric oxide, and connective tissue degrading metalloproteinase (MMP)enzymes. Due to its ability to induce metalloproteinases (MMPs), PGE2contributes to the breakdown of cartilage ECM. In addition, PGE₂promotes bone resorption and osteophyte formation. PGE₂ sensitizesnociceptors on peripheral nerve endings, thereby contributing to thedevelopment of inflammatory pain. PGE₂ levels are locally regulated bythe cyclooxygenase-2 (COX-2) enzyme. In pathologic conditions such asosteoarthritis, COX-2 expression is up-regulated with a concomitantincrease in PGE2 production.

TNF-α is a major mediator of inflammation and plays an important role intissue regeneration/expansion and destruction during inflammation. In anormal state, inflammation is well regulated by these factors. That is,after these factors cause inflammation with the concomitant induction ofimmune responses, their levels decrease to a normal state. However,deregulated TNF-α production causes chronic inflammation, which isdirectly associated with a variety of diseases such as arthritis.

While inflammation is a crucial immunological process necessary toresolve tissue injury or infection, the chronic release ofpro-inflammatory mediators like IL-1β and TNF-α can continue to induceproduction of additional inflammatory mediators. If levels do not returnto a normal state, the dysregulated production of TNF-α can potentiallylead to a detrimental pathophysiological process, includingosteoarthritis (OA).

TNF-α plays a key role in the initiation of the inflammatory process.TNF-α is produced by a variety of cells in the joint, namelychondrocytes, osteoblasts, cells in the synovial membrane, and residentimmune cells in the joint, or those that infiltrate the joint during theinflammatory response. Increased levels of TNF-α are detected insynovial fluid, synovial membrane, cartilage, and subchondral bone ofthose with osteoarthritis.

TNF-α along with IL-1β are capable of inducing Nuclear factor-kappa B(NF-κB), the master regulator of the inflammatory response. TNF-αinduces the production of PGE₂ by increasing the production of the keyenzymes involved in its synthesis, including COX-2, microsomal PGEsynthase (mPGES-1), and soluble Phospholipase A2 (sPLA2). Additionally,TNF-α induces the production of inducible nitric oxide synthase (iNOS)resulting in an increase in nitric oxide (NO) levels. The production ofother cytokines, including IL-6, IL-17 and IL-18 and the chemokine IL-8are positively modulated by TNF-α. In combination, the production ofthese pro-inflammatory mediators—prostaglandins, NO, cytokines andchemokines—ultimately results the in the breakdown of cartilageassociated with osteoarthritis.

TNF-α is capable of inhibiting the production of two key components ofthe extra cellular matrix—aggrecan and type II collagen. Further, TNF-αinduces the expression of aggrecanases ADAMTS4 and ADAMTS-5, enzymesthat degrade aggrecan. These two actions combined disrupt the normalbiochemical balance between synthesis and degradation of the cartilagematrix in the joint, ultimately resulting in cartilage degeneration.TNF-α has also been shown to play a role in mitochondrial dysfunction,decreased ATP production and apoptosis further contributing to cartilagedestruction. While TNF-α plays a central role in initiating theessential immune response to injury and infection, the deleteriouseffects that it triggers when dysregulated make TNF-α a target fordevelopment of inflammation management products.

The role of other tissues in the inflammation process is also wellestablished. Inflammation of the synovial membrane is now recognized tobe a key event in cartilage degradation in osteoarthritis, particularlyduring the early stages of the disease. Synovitis is characterized byactivation of resident macrophage-like cells and fibroblast-like cellsin the synovial membrane which leads to production of excessive amountsof pro-inflammatory mediators including TNF-α, IL-1 β, and PGE₂. Recentevidence suggests that synovial macrophages are the main source of thecytokines in the earliest stages of osteoarthritis and that they areimportant contributors to the cartilage damage in osteoarthritisthroughout the course of the disease. Cytokines also induce productionof PGE₂ and active metalloproteinases (MMPs). It is now well acceptedthat these mediators control the balance between ECM destruction andrepair, which has made these molecules preferred targets for therapeuticintervention. Other tissues in the joint such as the subchondral bonealso produce pro-inflammatory mediators that modulate joint health.

In addition to pro-inflammatory mediators such as cytokines andprostaglandins, reactive oxygen species (ROS) have also been implicatedin joint degeneration observed in osteoarthritis. Oxidative stressinduced by ROS such as nitric oxide and hydrogen peroxide has been shownto cause chondrocyte apoptosis and cartilage ECM breakdown. Moreover,ROS have been reported to activate signal transduction pathways thatlead to an increased production of pro-inflammatory mediators includingcytokines and prostaglandins. Studies in vitro have demonstrated alinkage between the pathways involved in the production of ROS andpro-inflammatory mediators. These studies support the notion that agentscapable of inhibiting both oxidative stress and inflammation pathwayswould be particularly useful in the modulation of inflammation.

The central role of COX-2 and PGE₂ in the pathophysiology ofosteoarthritis is reflected in the widespread use of selective COX-2inhibitors and a variety of non-selective non-steroidalanti-inflammatory drugs (NSAIDs) for the treatment of the disorder.However, prolonged administration of these drugs has adverse sideeffects, including gastrointestinal pathologies and disruption ofcartilage proteoglycan metabolism. Studies in human and animal modelshave demonstrated impaired bone healing and repair with the use of COXinhibitors. Therefore, there is a need for alternative treatments forthe management of inflammation that do not center on the use of NSAIDsto inhibit the production of PGE₂ and other pro-inflammatory mediators,including TNF-α.

Among the drugs developed thus far for targeting TNF-α are Infliximab (achimeric monoclonal antibody against human TNF), Adalimumab (a fullyhuman monoclonal antibody), Etanercept (a dimeric TNFRII (p75) fusionprotein linked to the Fc portion of human IgG), Golimumab, CDP571, andThalidomide. However, in addition to inhibiting the positive functionsof TNF-α, these drugs may elicit unwanted outcomes including lymphomadevelopment and infection. There is therefore a need for therapeuticagents that regulate the excessive reactive oxygen species generationand cell death which is induced by TNF-α without blocking the positivephysiological functions of TNF-α.

SUMMARY

In accordance with the purposes and benefits described herein, in oneaspect of the present disclosure a composition is provided comprising acombination of hydroxytyrosol and 3-O-acetyl-11-keto-β-boswellic acid.In embodiments, the hydroxytyrosol is sourced from an olive extract andthe 3-O-acetyl-11-keto-β-boswellic acid is sourced from a Boswelliaserrata extract. The composition may be formulated for oraladministration to a mammalian subject, which may be selected from thegroup consisting of a human, dog, cat, horse, camel, or cow. In otherembodiments, the composition may be formulated for oral administrationto an avian subject.

In embodiments, the composition formulated for oral administration to ahuman subject may comprise 3-O-acetyl-11-keto-β-boswellic acid in anamount of from about 0.67 to about 2.70 mg per kg bodyweight andhydroxytyrosol in an amount of from about 0.15 to about 2.50 mg per kgbodyweight. In embodiments, the composition formulated for oraladministration to a dog subject may comprise3-O-acetyl-11-keto-β-boswellic acid in an amount of from about 1.24 toabout 4.98 mg per kg bodyweight and hydroxytyrosol in an amount of fromabout 0.28 to about 4.60 mg per kg bodyweight.

In another aspect, the present disclosure provides a method of treating,repairing, or reducing damage to connective tissue caused by one or moreinflammatory mediators, comprising administering to a mammalian or aviansubject in need thereof an orally administrable composition comprising asynergistic combination of hydroxytyrosol and3-O-acetyl-11-keto-β-boswellic acid as described above.

In yet another aspect, the present disclosure provides a method ofreducing levels of one or more inflammatory mediators in connectivetissue, comprising administering to a mammalian or avian subject in needthereof an orally administrable composition comprising a synergisticcombination of hydroxytyrosol and 3-O-acetyl-11-keto-β-boswellic acid asdescribed above.

In the following description, there are shown and described embodimentsof the disclosed compositions and methods. As it should be realized, thedescribed compositions and methods are capable of other, differentembodiments and its several details are capable of modification invarious, obvious aspects all without departing from the subject matterset forth and described in the following claims. Accordingly, thedrawings and descriptions should be regarded as illustrative in natureand not as restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawing figures incorporated herein and forming a partof the specification, illustrate several aspects of the disclosedcompositions and together with the description serve to explain certainprinciples thereof. In the drawing:

FIG. 1 illustrates the effect of hydroxytyrosol and AKBA in certainconcentrations on TNF-α production in lipopolysaccharide-stimulated RAW264.7 mouse macrophage cells;

FIG. 2 illustrates the effect of hydroxytyrosol and AKBA in certainconcentrations on TNF-α production in lipopolysaccharide-stimulated RAW264.7 mouse macrophage cells; and

FIG. 3 illustrates the effect of hydroxytyrosol and AKBA in certainconcentrations on TNF-α production in lipopolysaccharide-stimulated RAW264.7 mouse macrophage cells.

Reference will now be made in detail to embodiments of the disclosedcompositions and associated methods, examples of which are illustratedin the accompanying drawing figures.

DETAILED DESCRIPTION

The present invention provides for methods comprising administration of(i) 3-O-acetyl-11-keto-β-boswellic acid (AKBA) and (ii) hydroxytyrosol,to a mammalian or avian subject. AKBA and hydroxytyrosol may beadministered together in one composition or dosage form, or they may beadministered separately. In certain embodiments, AKBA and hydroxytyrosolare administered together in one composition or dosage form, orseparately, within a period in which their therapeutic propertiesoverlap. In embodiments, the compositions are administered separatelywithin 1 hour. In other embodiments, the compositions are administeredseparately within 30 minutes. In still other embodiments, thecompositions are administered separately within 5 minutes.

The term “mammalian subject” is any mammal, including, but not limitedto humans, dogs, cats, horses, cows, and camels. The term “aviansubject” refers to birds.

Hydroxytyrosol is a type of phenolic phytochemical found in parts of theolive tree. Hydroxytyrosol has an IUPAC name of4-(2-Hydroxyethyl)-1,2-benzenediol and refers to a compound having thefollowing structure:

As used herein, hydroxytyrosol may be of either synthetic origin orobtainable from natural sources such as from products and by-productsderived from the olive tree by extraction and/or purification.Additionally, hydroxytyrosol may be administered in the form ofhydroxytyrosol-comprising extracts obtainable from products andby-products derived from the olive tree. Products and by-products ofolive trees encompass olives, olive tree leafs, olive pulps, olive oil,olive-derived vegetation water and olive oil dregs without being limitedthereto. Based on the extraction procedure the amount, and respectivelythe ratio of the hydroxytyrosol, can be easily adjusted by a personskilled in the art. In embodiments, the hydroxytyrosol is derived fromolives that may be obtained from conventional and commercially availablesources such as growers.

The hydroxytyrosol employed herein can be prepared by a number ofmethods known in the art. The olives may be processed by any suitablemeans to obtain the compositions described. For example, the olivesand/or olive leaves may be pressed to obtain a mixture including oliveoil, vegetation water and solid byproducts. The hydroxytyrosol may beobtained directly from the mixture or the mixture may be fractionatedand/or purified to obtain the hydroxytyrosol. The compositions may befractionated and/or purified by a number of methods known to the personskilled in the art. Examples of fractionating methods includepartitioning with an organic solvent, chromatography, high pressureliquid chromatography (HPLC), or the use of supercritical fluids.

Examples of references that deal with the extraction of hydroxytyrosolfrom olive leaves are WO02/18310 A1, US 2002/0198415 A1, WO2004/005228A1, U.S. Pat. No. 6,416,808 and US 2002/0058078 A1 which disclose amethod for acidic hydrolysis of olive vegetation water for 2 to 12months until at least 90% of the present oleuropein has been converted.A method of extraction of hydroxytyrosol from olives, olive pulps, oliveoil and oil mill waste water is described in U.S. Pat. No. 6,361,803 andWO01/45514 A1 and in US 2002/0004077 A1. EP 1 582 512 A1 describes anextraction of hydroxytyrosol from olive leaves. A method for obtaininghydroxytyrosol from the vegetation water of de-pitted olives isdisclosed in US 2004/0039066 A1 in paragraphs [0080]-[0091]. Similarlysuitable for use in the present invention are commercially availablehydroxytyrosol-containing olive extracts.

The oral bioavailability of a single 2.5 mg/kg dose of hydroxytyrosol inhuman subjects has been reported in the literature, with an observedpeak plasma concentration of 1.11±0.20 μmol/L. González-Santiago, etal., Pharmacological research, 61.4 (2010): 364-370. Dosage calculationscan be determined by those of skilled in the art by evaluating bodyweight, surface area, and species differences. Similarly, dosages forcross-species extrapolation can be calculated by one skilled in the artusing conventional dose conversion methods.

The typical dosage rate of hydroxytyrosol is about 0.001 mg/kg to about2.0 mg/kg. In some embodiments, the typical daily dosage is at least 0.1mg and up to 300 mg for human and non-human subjects. The daily dosagerefers to the total dosage administered in a 24-hour period.

According to some exemplary embodiments, hydroxytyrosol may beadministered at a dose of 0.15 to 2.50 mg per kg bodyweight of a humansubject (i.e. 9-250 mg for a 60 kg human subject).

According to some exemplary embodiments, hydroxytyrosol may beadministered at a dose of 0.28 to 4.60 mg per kg bodyweight of a dogsubject (i.e. 2.8-46 mg for a 10 kg dog subject).

Hydroxytyrosol may be administered at a frequency of one time per weekto five times daily. In embodiments, hydroxytyrosol is administered onceevery two days to three times daily. In alternative embodiments,hydroxytyrosol is administered one to two times daily. In still otherembodiments, hydroxytyrosol is administered once daily. Hydroxytyrosolmay be taken with or without the administration of food.

Phytochemicals extracted from Boswellia serrata have been reported to beactive in the treatment of numerous afflictions and maladies. The gumresin of Boswellia serrata has long been in use for the treatment ofrheumatoid arthritis and gout by the practitioners of Ayurvedicmedicines in the Indian system of medicine. Various extracts of the gumresin have shown potent anti-inflammatory and anti-atherogenic activityin laboratory animals. The biological activity of the extract has beenrelated to the components of the boswellic acid fraction.3-O-acetyl-11-keto-β-boswellic acid (AKBA) has been identified as themost active compound in Boswellia serrata extracts. Boswellia serrataextracts containing AKBA have been reported to inhibit 5-lipoxygenaseand matrix metalloproteinase-3 (MMP-3) in vitro, as described inWO2010/029578 A2. WO2010/029578 A2 similarly reports theanti-inflammatory efficacies of compositions comprising Boswelliaserrata extract selectively enriched in AKBA to 30% in vivo, includingsignificant reductions in the serum biomarkers TNF-α and IL-1 β.

The bioavailability of a single dose administration of 100 mg/kg dose ofBoswellia serrata extract standardized to 30% AKBA in rat serum has beenreported in the literature, with an observed peak serum concentration of2.0 micrograms/mL being reported. Sengupta, et al. Molecular andcellular biochemistry, 354.1-2 (2011): 189-197. Dosage calculations canbe determined by those of skilled in the art by evaluating body weight,surface area, and species differences. Similarly, dosages forcross-species extrapolation can be calculated by one skilled in the artusing conventional dose conversion methods.

The typical dosage rate of AKBA is about 0.01 mg/kg to about 10.0 mg/kg.In some embodiments, the typical daily dosage is at least 1 mg and up toabout 1 g for human and non-human subjects. The daily dosage refers tothe total dosage administered in a 24-hour period.

According to some exemplary embodiments, AKBA may be administered at adose of 0.67 to 2.70 mg per kg bodyweight of a human subject (i.e.40-162 mg for a 60 kg human subject).

According to some exemplary embodiments, AKBA may be administered at adose of 1.24 to 4.98 mg per kg bodyweight of a dog subject (i.e.12.4-49.8 mg for a 10 kg dog subject).

AKBA may be administered at a frequency of one time per week to fivetimes daily. In certain embodiments, AKBA is administered once every twodays to three times daily. In alternative embodiments, AKBA isadministered one to two times daily. In still other embodimentsembodiments, AKBA is administered once daily. AKBA may be taken with orwithout the administration of food.

In some embodiments, the combination of (i) hydroxytyrosol and (ii) AKBAdemonstrates synergy. Synergy refers to the effect wherein a combinationof two or more components provides a result which is greater than thesum of the effects produced by the agents when used alone. In certainembodiments, the result is statistically significant and greater thanthe additive effect. In some embodiments, the combination ofhydroxytyrosol and AKBA has a statistically significant, greater effectthan each component alone. In certain embodiments, the combination ofhydroxytyrosol and AKBA demonstrates synergy in one or more of thefollowing: preventing, treating, repairing or reducing damage toconnective tissues; reducing symptoms associated with damage toconnective tissue in an avian or mammalian subject; and reducing levelsof one or more inflammatory mediators in connective tissue.

The present invention provides a method of preventing, treating,repairing, reducing damage, or controlling inflammation of connectivetissues, protecting cartilage, or reducing symptoms associated withdamage to connective tissue in an avian or mammalian subject, comprisingadministering to the subject: (i) hydroxytyrosol and (ii) AKBA. The term“connective tissue” includes but not limited to cartilage, bone,synovium, ligament, meniscus, and tendon. In some embodiments, theadministration of (i) hydroxytyrosol and (ii) AKBA may prevent, treat,repair or reduce damage to connective tissues. The damage to connectivetissue may be a result of physical injury or may represent “wear andtear” from continual use, weight and age, for example, fromosteoarthritis. Damage to connective tissue may also result from diseasesuch as rheumatoid arthritis, synovial disorders, infection relatedrheumatic diseases and inflammatory connective tissue disorders. In someembodiments, the administration of (i) hydroxytyrosol and (ii) AKBA mayreduce symptoms associated with damage to connective tissue in an avianor mammalian subject. Symptoms associated with damage to connectivetissue include, but are not limited to: pain, discomfort, pressure,inflammation, stiffness and/or swelling.

The present invention also provides a method of reducing levels of oneor more inflammatory mediators in connective tissue, comprisingadministering to an avian or mammalian subject: (i) hydroxytyrosol and(ii) AKBA. The inflammatory mediators include, but are not limited totumor necrosis factor-α (TNF-α), prostaglandins such as prostaglandin E₂(PGE₂), cytokines such as interleukin-1β (IL-1β) and, chemokines,leukotrienes, nitric oxide, and reactive oxygen species.

The administration of hydroxytyrosol and AKBA may also be useful fortreating, preventing, and reducing damage or reducing symptomsassociated with conditions affecting the cardiovascular system, nervoussystem, musculoskeletal system and gastrointestinal system. In oneaspect, the present disclosure provides compositions and methods forpreventing and/or reducing an inflammatory response and/or inflammationin a subject. In one aspect, the present disclosure providescompositions and methods for managing inflammatory disorders orgenerally reducing inflammatory burden of a human or non-human animal.Accordingly, in one embodiment, the present invention provides a methodof preventing and/or reducing an inflammatory response and/orinflammation in one or more tissues, the method including delivering tothe one or more tissues the compositions of the present invention.

The present invention also provides for an orally administrablecomposition comprising: (i) hydroxytyrosol and (ii) AKBA. The orallyadministrable composition is any dosage form which can be administeredorally, such as, but not limited to: a capsule, a tablet, a powder thatcan be dispersed in a beverage, a paste, in pelletized form, a liquidsuch as a solution, suspension, or emulsion, a soft gel/chew capsule, achewable bar or other convenient dosage form such as oral liquid in acapsule, as known in the art.

The orally administrable composition may contain one or more non-activepharmaceutical ingredients (also known generally herein as“excipients”). Non-active ingredients, for example, serve to solubilize,suspend, thicken, dilute, emulsify, stabilize, preserve, protect, color,flavor, and fashion the active ingredients into an applicable andefficacious preparation that is safe, convenient, and otherwiseacceptable for use. The excipients may be pharmaceutically acceptableexcipients. Examples of classes of pharmaceutically acceptableexcipients include lubricants, buffering agents, stabilizers, blowingagents, pigments, coloring agents, flavoring agents, fillers, bulkingagents, fragrances, release modifiers, adjuvants, plasticizers, flowaccelerators, mold release agents, polyols, granulating agents,diluents, binders, buffers, absorbents, glidants, adhesives,anti-adherents, acidulants, softeners, resins, demulcents, solvents,surfactants, emulsifiers, elastomers and mixtures thereof.

The orally administrable compositions may further comprise one or moreactive ingredients. For example, the compositions may further compriseone or more drugs or nutritional supplements. In some embodiments, thecompositions may further comprise compounds which are beneficial toconnective tissue. Example include, but are not limited toglycosaminoglycans such as chondroitin, aminosugars such as glucosamine,methylsulfonylmethane (MSM), collagen (including collagen type II),green tea extracts, scutellaria extracts, acacia extracts, turmericextracts, curcumin, cetyl myristoleate complex (CMO) and egg shellmembrane.

All references cited herein are incorporated by reference in theirentirety.

EXAMPLES Example 1: Effect of Hydroxytyrosol and AKBA on TNF-αProduction in Lipopolysaccharide (LPS) Stimulated RAW 264.7 MouseMacrophage Cells

RAW 264.7 mouse macrophage cells were pre-treated with 60 nM, 160 nM, or1 μM hydroxytyrosol (HT) (98% purity, Sigma-Aldrich, St. Louis, Mo.)alone, 0.28 μg/mL, 0.56 μg/mL or 1.124 μg/mL AKBA (administered as5-LOXIN®, standardized to 30% AKBA, PLT Health Solutions, Inc.) alone,or each of the three concentrations of HT combined with each of thethree concentrations of AKBA for 24 hours. Cells were then stimulatedfor an additional 24 hours with 1 μg/mL lipopolysaccharide (LPS). LPS isan endotoxin in the bacterial cell wall capable of inducing aninflammatory response which includes an increased production of TNF-α.Cellular supernatants were analyzed for TNF-α production. Statisticalcomparisons were made using one-way analysis of variance (ANOVA) andTukey post-hoc analysis was performed where differences of P<0.05 wereconsidered significant. Data is presented as the mean+/−1 SD.

Statistically significant greater reductions in the levels of TNF-α wereobserved when each of the three concentrations of HT were combined withAKBA compared to the reduction by either agent alone. The combination of60 nM HT with either 0.28 μg/mL, 0.56 μg/mL, or 1.124 μg/mL AKBAresulted in a greater reduction of TNF-α production than either HT(P<0.001) or AKBA (P<0.001) alone (FIG. 1). Statistical significance wasreached in the reduction of TNF-α in cells treated with 160 nM HT incombination with 0.56 μg/mL AKBA compared with either HT (P<0.001) orAKBA (P=0.001) alone (FIG. 2). The treatment of cells with 1 μM HT andeither 0.28 μg/mL, 0.56 μg/mL, or 1.124 μg/mL AKBA also resulted instatistical significant reductions compared to HT alone (P<0.001,P=0.002 and P<0.001, respectively) and AKBA alone (P<0.001, P=0.02 andP=0.004, respectively) (FIG. 3).

What is claimed:
 1. A composition, comprising hydroxytyrosol and3-O-acetyl-11-keto-β-boswellic acid provided in an amount effective toprevent or reduce an inflammatory response in connective tissue of asubject in need thereof.
 2. The composition of claim 1, wherein thehydroxytyrosol is sourced from an olive extract.
 3. The composition ofclaim 1, wherein the 3-O-acetyl-11-keto-β-boswellic acid is sourced froma Boswellia serrata extract.
 4. The composition of claim 1, formulatedfor oral administration to a mammalian subject or an avian subject. 5.The composition of claim 4, wherein the mammalian subject is selectedfrom the group consisting of a human, dog, cat, horse, camel, or cow. 6.The composition of claim 5, wherein the composition formulated for oraladministration to the human subject comprises3-O-acetyl-11-keto-β-boswellic acid in an amount of from about 0.8 toabout 2.50 mg per kg bodyweight.
 7. The composition of claim 5, whereinthe composition formulated for oral administration to the dog subjectcomprises 3-O-acetyl-11-keto-β-boswellic acid in an amount of from about1.4 to about 4.50 mg per kg bodyweight.
 8. The composition of claim 5,wherein the composition formulated for oral administration to the humansubject comprises hydroxytyrosol in an amount of from about 0.2 to about2 mg per kg bodyweight.
 9. The composition of claim 5, wherein thecomposition formulated for oral administration to the dog subjectcomprises hydroxytyrosol in an amount of from about 0.4 to about 4 mgper kg bodyweight.
 10. The composition of claim 1, wherein theinflammatory response is mediated by one or more of tumor necrosisfactor alpha, prostaglandin E₂, cyclooxygenase-2, and nitric oxide. 11.A method of treating, repairing, or reducing damage to connective tissuecaused by one or more inflammatory mediators, comprising administeringto a subject in need thereof an orally administrable compositioncomprising a synergistic combination of hydroxytyrosol and3-O-acetyl-11-keto-β-boswellic acid.
 12. The method of claim 11, whereinthe subject is a mammalian subject or an avian subject.
 13. The methodof claim 11, including deriving the hydroxytyrosol from an oliveextract.
 14. The method of claim 11, including deriving the3-O-acetyl-11-keto-β-boswellic acid from a Boswellia serrata extract.15. The method of claim 11, including providing the orally administrablecomposition formulated for a human subject comprising3-O-acetyl-11-keto-β-boswellic acid in an amount of from about 0.67 toabout 2.70 mg per kg bodyweight.
 16. The method of claim 15, includingproviding the orally administrable composition formulated for a humansubject comprising 3-O-acetyl-11-keto-β-boswellic acid in an amount offrom about 0.80 to about 2.50 mg per kg bodyweight.
 17. The method ofclaim 11, including providing the orally administrable compositionformulated for a dog subject comprising 3-O-acetyl-11-keto-β-boswellicacid in an amount of from about 1.24 to about 4.98 mg per kg bodyweight.18. The method of claim 17, including providing the orally administrablecomposition formulated for a dog subject comprising3-O-acetyl-11-keto-β-boswellic acid in an amount of from about 1.4 toabout 4.50 mg per kg bodyweight.
 19. The method of claim 11, includingproviding the orally administrable composition formulated for a humansubject comprising hydroxytyrosol in an amount of from about 0.15 toabout 2.50 mg per kg bodyweight.
 20. The method of claim 19, includingproviding the orally administrable composition formulated for a humansubject comprising hydroxytyrosol in an amount of from about 0.2 toabout 2 mg per kg bodyweight.
 21. The method of claim 11, includingproviding the orally administrable composition formulated for a dogsubject comprising hydroxytyrosol in an amount of from about 0.28 toabout 4.60 mg per kg bodyweight.
 22. The method of claim 21, includingproviding the orally administrable composition formulated for a dogsubject comprising hydroxytyrosol in an amount of from about 0.4 toabout 4 mg per kg bodyweight.
 23. The method of claim 11, includingadministering the hydroxytyrosol and the 3-O-acetyl-11-keto-β-boswellicacid to the subject together as a single composition or separately. 24.The method of claim 23, including administering the hydroxytyrosol andthe 3-O-acetyl-11-keto-β-boswellic acid to the subject separately withina one-hour time frame.
 25. The method of claim 24, includingadministering the hydroxytyrosol and the 3-O-acetyl-11-keto-β-boswellicacid to the subject separately within a 30-minute time frame.
 26. Themethod of claim 25, including administering the hydroxytyrosol and the3-O-acetyl-11-keto-β-boswellic acid to the subject separately within a5-minute time frame.